L-Carnitine (LC), an essential co-factor for the metabolism of long chain fatty acids, is synthesized endogenously & obtained from dietary sources. Dietary sources are important for normal homeostasis and, especially, in deficiency states. The mechanism of LC intestinal absorption is not clear. Specific carrier-mediated & non-specific simple passive diffusion have been proposed but this issue remains unresolved. To address this issue a study using the human-derived intestinal epithelial cells. Caco-2 to determine the mechanism of LC intestinal uptake has been initiated. Preliminary results indicate involvement of a specialized carrier-mediated system, as shown by significant (less than 0.05) inhibition of 3H-LC uptake by high concentration unlabeled LC & its structural analogues, D-carnitine, acetyl- DL-carnitine & (3-carboxypropyl)trimethyl-ammonium chloride. The goal of this proposal is to expand these preliminary findings. Specifically to; A. Further investigate the mechanisms of LC uptake using Caco-2 cells, by examining 1) LC uptake as a function of time & its structural analogues group specific reagents, metabolic & membrane uptake inhibitors, &5) the effect of LC levels on the substrate uptake by Caco-2 cells. B. Examine possible role of intracellular signaling mechanisms in regulating LC uptake. The role of protein kinase A (PKA), C (PKC) & G (PKG) mediated pathways in regulating LC uptake will be examined using a variety of specific stimulators & inhibitors of these intracellular second messenger pathways. Examination of effect of kinase stimulation with & without the presence of phosphatase inhibitors will also be done. C. To use the Xenopus laevis oocyte expression system to functionally express the LC transporter & to determine the mRNA fraction that expresses the highest uptake activity. D. To construct a cDNA library from the mRNA fraction with the highest activity in a plasmid expression vector & to screen to determine the positive clone(s). E. To determine the sequence of the cDNA of the positive clone(s) & to predict the amino acid sequence of the protein. Subsequent analysis of tahe deduced amino acid sequence to determine the hydrophobic secondary structures, allowing athe identification of the apparent three-dimensional configuration of the carrier and F. Using probes generated from the cloned cDNA in Northern blot analyses to determine the distribution of expression of mRNA encoding the transporter in other tissues. These studies will lead to an understanding of the mechanism(s) of the L- carnitine intestinal absorption process its regulation and the molecular identity of its transporter which may help in clinical use of L-carnitine in deficiency or insufficiency states as well s in normal homeostasis.